Structural differences within the loop E motif imply alternative mechanisms of viroid processing
Abstract
Viroids replicate via a rolling circle mechanism, and cleavage/ligation requires extensive rearrangement of the highly base-paired native structure. For Potato spindle tuber viroid (PSTVd), the switch from cleavage to ligation is driven by the change from a multibranched tetraloop structure to a loop E conformation. Here we present evidence that processing of Citrus viroid III (CVd-III), a member of a related group of viroids that also replicate in the nucleus, may proceed via a distinct pathway. Chemical probing of PSTVd and CVd-III miniRNAs with DMS and CMCT revealed that the loop E motifs of these two viroids have quite different tertiary structures. As shown by temperature gradient gel electrophoresis, the presence of two likely Watson–Crick GC pairs results in a significant overall stabilization of the CVd-III loop E-like motif. Unlike PSTVd, the upper strand of the CVd-III loop E-like motif cannot fold into a GNRA tetraloop, and comparison of suboptimal structures indicates that the initial cleavage event could occur on the 5′ side of the only GU wobble pair in a helix involving a nearby pair of inverted repeats. According to our model, rearrangement of 3′ sequences into a hairpin stem containing an identical arrangement of GC, GU, and CG base pairs and a second cleavage event is followed by formation of loop E, which serves to align the 5′ and 3′ termini of the CVd-III monomer prior to ligation. Because ligation would occur within loop E itself, stabilization of this motif may be needed to hold the 5′ and 3′ termini of CVd-III in position for the host ligase.
Keywords
Footnotes
-
Reprint requests to: Robert A. Owens, Molecular Plant Pathology Laboratory, Room 118 Building 004, Beltsville Agricultural Research Center, Beltsville, MD 20705, USA; e-mail: robert.a.owens{at}ars.usda.gov; fax: (301) 504-5449.
-
Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.452307.
-
- Received January 10, 2007.
- Accepted March 10, 2007.
- Copyright © 2007 RNA Society